Soil Biol. Biochem. Vol. 25, No. 6, pp. 715-721,
1993 Printed in Great Britain
0038-0717/93 $6.00 + 0.00
Pergamon Press Ltd
INFLUENCE OF ACID SOIL ON NODULATION AND
INTERSTRAIN COMPETITIVENESS IN RELATION TO
TANNIN CONCENTRATIONS IN SEEDS AND ROOTS OF
PHASEOLUS VULGARIS
A. B. WOLFF, * P. W. SINGLETON, M. SIDIRELLI * and B. B. BOHLOOL
University of Hawaii, NifFAL Project, 1000 Holomua Avenue, Paia, Maui, HI 96779-9744, U.S.A.
(Accepted 20 December 1992)
Summary-The relationship between acid soils and (1) early nodulation in common beans, (2)
interstrain competitiveness of Rhizobium leguminosarum bv. phaseoli strains and (3) tannin concentration
in roots, were evaluated in two experiments. Ten Phaseolus vulgaris cultivars of different seed tannin
content were grown in pots in a greenhouse in soil at pH 4.5 or 5.5, inoculated with equal amounts of
strains CIAT 899 and KIM5. At pH 5.5, on all cultivars strain KIM5 occupied a greater proportion of
nodules than strain CIAT 899. This was reversed at pH 4.5. Nodule number and nodule weight, 21
days after inoculation, were strongly reduced but root tannin concentrations doubled at pH 4.5. Nodule
numbers were negatively correlated with tannin concentration in roots at both pH values. Seed tannin
content was negatively correlated with early nodulation, but this was statistically significant only at pH
5.5. Nodulation of cultivars with high seed tannin contents was generally more acid tolerant than of
those with low seed tannin content. In a second experiment, six Phaseolus vulgaris cultivars were
planted at two field sites with acid soil and one site with neutral soil. In the field, as in the pots, nodule
numbers at early stages of plant development were reduced by 30-50% in the acid soils compared to
the neutral soil. In contrast, root tannin content in acid soils was twice that in the neutral soil. There
were significant cultivar and cultivar by location effects.
Strains of rhizobia were tested for tolerance to bean seed tannin extracts, to seed diffusates and for
survival on seed surfaces. The Rhizobium strains were sensitive to tannin extracts, with obvious strain
differences. Sensitivity of most strains was greater at acidic pH. Seed diffusates, in contrast, did not
inhibit growth of most strains.
INTRODUCTION
Various types of common bean are the main sources of
vegetable protein for people in Latin America. They are
cultivated mainly by small-scale farmers on marginal,
frequently acid soils (Vargas and Graham, 1989). An
increased understanding of the mechanisms involved in pH
tolerance of plants could help in breeding or selecting
plants and rhizobia that are better adapted to acid soils.
The formation of nitrogen-fixing nodules on roots of
legumes is influenced by many environmental factors
(Kosslak and Bohlool, 1985; Abaidoo et al., 1990). In
particular, nodule formation is sensitive to low soil pH
(and associated factors, e.g. Al and Mn toxicity, Ca deficiency) and high soil temperatures. The precise effects of
pH and the other stress factors on nodulation of legumes,
however, remain poorly understood. Bean seeds contain
components commonly known as antinutritional factors,
one of the most important being tannins. These
polyphenolic substances repel predators, inhibit enzymes,
form insoluble, indigestible complexes with proteins,
carbohydrates, Ca, Mg and Fe. They also inhibit growth of
rhizobia and
*Present address: ICMEA, Ellinikis Dimokratias 24,
GR-65201 Kavala, Greece.
715
nodulation of groundnuts and common beans (for a
review see Muthukumar et al., 1985). Tannin production
in plants can be stimulated by environmental stress, such
as low soil fertility (Pankhurst and Jones, 1979) which is
common in the tropics.
We have investigated the effect of acidity on early
nodulation, interstrain competitiveness, plant growth and
tannin concentration in the roots of different bean
cultivars. Correlations between either (a) early nodulation
or (b) inoculant strain competitiveness and tannin content
of seeds or roots are demonstrated. To test the hypothesis
that the reduction of nodule numbers at high tannin
concentrations in roots is due to their toxicity for rhizobia,
the response of R.. leguminosarum bv. phaseoli strains to
tannin extracts, to seed diffusates and on seed surfaces
was observed.
MATERIALS AND METHODS
Inoculum strains and seed sources
Rhizobium leguminosarum bv. phaseoli strains and
Phaseolus vulgaris cultivars were obtained from the
strain and germplasm collections of NifTAL-Project and
the Centro Internacional de Agricultura Tropical,
(CIAT), Cali, Colombia.
2
A. B. WOLFF
et
al.
Plants were harvested at 22 days and shoot dry
weight, root dry weight, nodule numbers, nodule
weight, root tannins and per cent nodule occupancy
Experiments were conducted at three field sites by strain CIAT 899 or KIM5 or double strain
at elevations of 110, 290 and 660 in on the island of
infection were analyzed.
Maui, Hawaii and are referred to herein as
Hamakuapoko, Kuiaha and Haleakala, respectively. Strain identification
The soil at the Hamakuapoko site was a fine,
Nodule occupancy by inoculant strain was deterKaolinitic, isohyperthermic, Typic Haplustoll mined by indirect enzyme-linked immunosorbent as(annual rainfall: 1130 mm; neutral, highly fertile, says (Wolff et al., 1991).
large amounts of soil N). The soil at the Kuiaha site
was a clayey, ferritic, isohyperthermic Humoxic Tannin assay
Trophohumult (annual rainfall: 2110 mm; pH 4.5,
moderate soil N) and that at the Haleakala site was a Tannins in seeds and roots were determined following the
clayey, oxidic, isohyperthermic Humoxic Tropohu- method of Deshpande and Cheryan (1985). Dry seeds or
mult (annual rainfall: 1979 mm; pH 3.9-4.8; moder- roots were ground and a 100 mg sample was extracted
ate soil N). Except for Hamakuapoko, the sites were with 1.5 ml 1% HCl in methanol for 10 min. The samples
free of indigenous bean infecting rhizobia (Woomer were then centrifuged at 1500g and aliquots were
et al., 1988). To minimize differences in soil immediately analyzed for tannin using the Vanillin Assay
environment, soils at all sites were amended before (Broadhurst and Jones, 1978).
the start of experiments by applying nutrients Reaction of Rhizobium strains to bean tannins
(except N) in recommended amounts (George et al.,
1987). Nutrients added (kg ha-') were 300P; 200K; Tannins were extracted in methanol-water (70:30 v : v),
with 1 % formic acid, from the testa of bean seeds and the
50Mg; IOZn; 0.5B; 0.5Mo.
For pot experiments soil from the Kuiaha site sensitivity of R.. leguminosarum bv. phaseoli strains to the
was sieved (< 5 mm). Half of the soil was amended crude bean tannin extracts was assayed as growth
with Ca(OH) 2 to raise the initial pH from 4.5 to 5.5. inhibition on agar plates at pH 5, 6 and 7 (Wolff et al.,
Soils were mixed with fertilizers (mg kg- ') 200P; 1991).
25Mg; 300K and micro-nutrients 0.2Mo; 1.75B;
Toxicity of seed exudates
2.5Zn; 7.5Fe.
Site characteristics and amendements
Inoculum preparation
Peat-based inocula of R.. leguminosarum bv. phase oli strains KIM5 (Ta1943), CIAT 632 (Tal 1383) and
CIAT 899 (Tal 1797) were prepared as described by
Somasegaran and Bohlool (1990).
Experimental design
Field experiments (plot size 40 m 2) were made
using a complete random block design, with four
replications, at Hamakuapoko, Kuiaha and
Haleakala. Six P. vulgaris cultivars (APN 84, BAT
271, Chingo, DOR 42, RAB 39, XAN 40) were
planted (20 seeds m- ' row) and each seed was
inoculated at planting time with an aqueous peatinoculant suspension of the R.. leguminosarum bv.
phaseoli strains CIAT 899 + CIAT 632 (1:1 mixture
with 108 rhizobia per seed). Plants were harvested
after 23 days and shoot dry weight, root dry weight,
nodule numbers and root tannin content were
determined.
Pot experiments were done in the greenhouse at
the Haleakala research station. Plastic pots (3 1.)
were used for the experiments and each pot
contained 2.6 kg of soil at field capacity (320 g H 2O
kg- ' soil) at either pH 4.5 or 5.5. Ten bean cultivars
(APN 42, Bolita 42; BAT 1449; Cuarentemo de
Honduras; Aqua Caliente 92; Guerrero 924; MCD
2004; Red Cloud; Rio Tibagi; Talamanca) were
planted with six replications each and inoculated
with a double strain mixture of R. leguminosarum bv.
phaseoli strains CIAT 899 and KIM5 as described
above. Uninoculated controls were also included.
Toxicity of bean seed exudates on R. leguminosarum bv.
phaseoli strains was tested by the method of Materon and
Weaver (1984). Cultures were grown in 20E medium
(Streit et al., 1991) on a rotary shaker. Log phase cultures
were used to inoculate melted 20E agar that had been
cooled to 45°C. The agar contained approx. 5 x 10 4 cells
ml - ' and 15 ml was added to standard plastic Petri dishes
and allowed to solidify. Seeds of P. vulgaris cultivars
were autoclaved and than placed onto the surface of the
solidified agar and incubated at 28 °C. The degree of
toxicity was measured after 1-3 days as the diameter of
the zone of growth inhibition around each seed.
Survival of strains on the seed surface
Seeds were surface sterilized for 1 min in 70% ethanol,
followed by a 5 min exposure to 3% sodium hypochlorite
and then four rinses in sterile water. The surface
sterilized seeds were soaked in wash cultures of R..
leguminosarum bv. phaseoli strains (10'° cells ml - ') for 10
min. After this inoculation, the seeds were placed on
water agar in Petri dishes and kept at 20°C in the dark.
Three replicate samples (10 seeds each) were taken.
Counts of Rhizobium on seeds were done by plating after
serial dilution and expressed as colony forming units per
seed (cfu seed - ').
Statistical analysis
Analysis of variance, Duncan multiple range tests
and regression analysis were done by using
Nodulation in acid
71
soils
7
STATGRAPHICS program from STSC, Inc.,
In the pot experiments, in addition to the pH effect,
there was also an effect of cultivar and a cultivar x pH
Rockville, Md, U.S.A.
interaction on most of the tested variables. Cultivars
differed significantly in early nodulation. Differences
between cultivars for root tannin content were small
The nodule numbers, shoot and root dry weights, root
although the cultivars varied widely in seed tannin
tannin content and nodule occupancy by inoculant strains
content (Fig. 1).
of 10 bean cultivars grown in pots of soil amended to two
In the field experiments, significant location, cultivar
different pH values, are presented in Table 1. There was a
and cultivar x location effects on all variables were
significant (P < 0.05) effect of pH on all symbiotic
found (Table 2). Highest nodule numbers, shoot and root
variables tested, such as nodule numbers, nodule weight
dry weights but lowest root tannin concentration were
and inoculant strain competitiveness. Nodule weight at pH found at the NifTAL field site at Hamakuapoko. Early
4.5 was reduced to half that at pH 5.5, while nodule
nodulation (NN) measurements in the acid soils of
numbers were reduced by an average of 25%. In contrast,
Kuiaha was 50% and at Haleakala 30% of that in the
tannin content in roots of plants at low pH was nearly
neutral field site of Hamakuapoko. In contrast, root
twice as high as in roots at the higher pH value. Interstrain tannin content at Kuiaha and Haleakala was about twice
competitiveness was also influenced by pH. At pH 5.5.
as high as at Hamakuapoko. Beans at Haleakala had the
strain KIM5 occupied about 70% of nodules and CIAT
highest tannin concentration but lowest nodule numbers
899 only 15%. At pH 4.5 strain CIAT 899 (50%) was
(Fig. 2).
superior to KIM5 (35%). On most cultivars more nodules
The bean cultivars used in this study varied widely in
were occupied by both strains at pH 4.5 than at pH 5.5.
tannin content of the seeds (Fig. 1). There was no
Cultivar differences in nodule occupancy by inoculant
significant correlation between tannins in seeds and
strain were small but were more pronounced at pH 5.5.
tannins in roots, either in the pot or the field experiments
At 21 days from planting there were no significant (Tables 1 and 2). Table 3 shows the correlations found
differences between inoculated and uninoculated control between the tannins in seeds or roots and the symbiotic
plants in shoot and root dry weights or root tannin variables. Nodule number and nodule dry weights from
content. No nodules were observed on the control plants. the pot experiments were both negatively correlated with
the tannin content of the roots. This tendency was partly
confirmed by the field experiments, in which pooled
RESULTS
data from the three
4
A. B. WOLFF et
locations showed that nodule numbers and root tannins
were inversely related (Fig. 2). However, there was no
significant correlation between nodule numbers and root
tannins when the field sites were analyzed individually.
Interstrain competitiveness
al.
was also related to root tannin content (NO, Table 3),
but was significant only at pH 4.5 in pots.
Seed tannin content was also negatively correlated
with nodule numbers, but this correlation was significant
only at pH 5.5 in pots (Table 3).
Sensitivity of the rhizobial strains to bean tannin
extract increased at low pH [Table 4(a)]. There were
obvious strain differences in relation to the pH effect.
While the strains H2C and KIM5 were strongly inhibited
at all pH values, the sensitivity of strains CIAT 151 and
CIAT 899 increased markedly at pH 5. Of all the R.
leguminosarum bv. phaseoli strains KIM5 and H2C
were the most sensitive at all pH values tested. Strain
CIAT 899 was relatively tolerant of tannins.
The effects of seed diffusates on the Rhizobium
strains is shown in Table 4. Strain H2C was sensitive to
the bean seed diffusates. None of the other strains tested
was inhibited by seed diffusates from any cultivar at that
time. Growth of strains CIAT 899 and CIAT 151 was in
many cases promoted in a zone around the seeds. The
effect was strongest for strain CIAT 899.
To investigate the effect of seed tannins on the
survival of R. leguminosarum bv. phaseoli strains in
vivo, seeds from cultivars of different tannin content
were selected. Although there was a significant
inhibition by the tannins on the growth of the R.
leguminosarum bv. phaseoli strains in vitro, no
inhibition of growth on the seed surface was detected
5
[Table 4(c)]. Cell number per seed increased approximately by 1 log unit over a period of 4 days and the
trend was similar for all cultivars. No significant strain
differences were observed.
DISCUSSION
High nodule numbers at early stages of plant
development have been shown to be positively correlated with total plant N late in the growth cycle and
SBB 25/6F
hence it could be considered as an important factor in
bean breeding programs for enhanced biological
nitrogen fixation (BNF) (CIAT, 1989). The world's
main bean producing areas are in the tropics, where a
large proportion of arable land is acidic, a major factor
limiting nodulation and N2 fixation in common beans
(Vargas and Graham, 1989). Additionally, symbiotic
legumes are often more sensitive to unfavorable soil
conditions than their nitrogen fertilized counterparts
(Piha and Munns, 1987). Although an understanding of
the plant-microbe interaction under stress is of great
economical importance, knowledge at the physiological
and genetic level is very limited.
In our pot culture trials and field experiments, early
nodulation was strongly affected by soil pH and cultivar
(cf. Vargas and Graham, 1989). Cultivars differed
widely in acid tolerance with significant pH x cultivar
and cultivar x location interactions. None of the
cultivars proved to be most successful in early
nodulation at all locations or all pH values tested (cf.
CIAT, 1989).
Acid sensitivity of nodulation was not related to
reductions in either shoot or root dry weight. Interestingly, nodule weight was more affected by soil pH
than were nodule numbers for all cultivars. This
confirms the observations of Evans et al. (1980) who
found that nodulation can be 10 times more sensitive
6
A. B. WOLFF
to acidity than the growth of Rhizobium or the legume
root alone.
Nodule occupancy by strain KIM5 or CIAT 899 was
influenced by bean cultivar. Such effects in the
greenhouse have already been described by DeOliveira
and Graham (1990). In field experiments, however,
cultivar effects on nodule occupancy by inoculant strain
is often less pronounced (Wolff et al., 1991). Interstrain
competitiveness is known to be a stable factor of a given
strain (Kosslak and Bohlool, 1985). Strain KIM5 is
known to be more competitive than CIAT 899 under
various conditions (Somasegaran and Bohlool, 1990). In
a low pH soil, however, per cent nodule occupancy by
CIAT 899 was higher than by KIM5. A few reports for
R. leguminosarum bv. phaseoli show that interstrain
competitiveness can be altered by abiotic stress, e.g. low
soil pH or high soil temperatures. Effects of acidity were
described between the R.. leguminosarum bv.
phaseoli subgroups I and II (subgroup II is now
classified as R. tropici). Under moderate pH
conditions, R. tropici strains are less competitive than
type I strains (Martinez-Romero et al., 1991). Whether
the enhanced inter-strain competitiveness at low pH is a
general feature of R. tropici strains needs further
investigation. R. tropici strains are known to be acid
tolerant in vitro (Martinez-Romero et al., 1991). In the
double-strain inoculants used in this study, strain CIAT
899 was acid tolerant while KIM5 was more sensitive
(Wolff et al., 1991). Under the controlled conditions of
the pot experiment, the change in interstrain competitiveness at pH 4.5 could be due to their differential
sensitivity to low pH. However no significiant differences were found for strain survival rates in an acid
et al.
soil (Streit et al., 1991). Also in the presence of an
indigenous Rhizobium population, competitiveness of
both strains decreased to the same extent in acid soils
(Wolff et al., 1991). Vargas and Graham (1989) found
no significant differences in the number of the R..
tropici strain CIAT 899 and the R.. leguminosarum
bv. phaseoli group I strain CIAT 632 (acid sensitive) in
the rhizosphere of beans at pH 4.5. Therefore, it appears
doubtful whether differences in acid tolerance of
Rhizobium strains in vitro can predict in vivo
symbiotic response.
Tannins are known to inhibit nodulation in beans
and other legumes when applied to the soil (Blum and
Rice, 1969; Muthukumar et al., 1985), but there are no
known reports on the effect of tannins in the seed coat or
roots on nodulation in beans. Our results show that
tannins of both origins can affect nodule numbers during
early stages of plant development. Nodule occupancy by
the tannin-sensitive strain KIM5 was negatively
correlated to root tannin content of the cultivars. With
CIAT 899 (tannin-tolerant) the correlation was positive.
Tannins are more actively bound at low pH (Haslem,
1989) and the sensitivity of the Rhizobium strains to
tannins under acid conditions may be related to binding.
Nodulation of cultivars whose root tannin content
increased strongly when grown in acid soils, tended to
be acid sensitive. Nodulation of cultivars with high seed
tannin content was in general more tolerant of low pH.
The correlations between tannin content and nodule
numbers/strain competitiveness suggest the possibility
that plant defense mechanisms, triggered in response to
abiotic stress, may be partly responsible for the poor
nodulation in acid soils in the tropics.
The mechanism by which tannins influence nodulation
is unknown, but it can be assumed that inhibition of
growth of Rhizobia (Pankhurst and Jones, 1979) may be
important. We also found growth inhibition of the tested
strains by tannin extracts in vitro. But our experiments
fail to show inhibition on seed surfaces. Other unknown
factors may have also enhanced the effects of seed
tannins in this experiment and confound the difficulty in
correlating in vivo behavior of a strain and its tannin
tolerance shown in vitro. Therefore the mechanism by
which tannins lead to a reduction in nodulation remains
unclear. However it is noteworthy that strains which
were tolerant to tannins were also tolerant to acidity
(Wolff et al., 1991).
Environmental stress limits the RhizobiumPhaseolus
symbiosis by reducing nodulation and nitrogen fixation
and increasing nodule senescence. Broad ranges of
cultivar and strain responses to abiotic stress factors
have been described. For a better understanding of these
processes more studies, at the physiological or genetic
level, of host-strain interaction under stress
conditions are necessary.
Nodulation in acid soils
7
Acknowledgements-A. B. Wolff wishes to thank the OrganHaslem E. (1989) Plant Polyphenols. Vegetable Tannins
ization of Economic Cooperation and Development for a
Revisited. Cambridge Univ. Press, Cambridge.
fellowship held at Hawaii under the OECD Project on
Kosslak R. M. and Bohool B. B. (1985) Influence of
Biological Resource Management.
environmental factors on interstrain competition in RhiWe thank the NifTAL-Project, especially G. Haines and
zobium japonicum. Applied and Environmental MicroK. Keane for assistance in the greenhouse and the field and
biology 49, 1128-1133.
D. Werner, University of Marburg, N. Cebel, Soil and
Martinez-Romero E., Segovia L., Mercante F. M., Franco
Fertilizer Research Institute, Ankara, and C. Montealegre,
A. A., Graham P. and Pardo M. A. (1991) Rhizobium
Centro Internacional de Agricultura Tropical, Cali, for
tropici, a novel species nodulating Phaseolus vulgaris L.
helpful discussions.
Beans and Leucaena sp. trees. International Journal of
Systematic Bacteriology 41, 417-426.
Materon L. A. and Weaver R. W. (1984) Survival of
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